Abstract Infections due to resistant Gram+ organisms are on the rise, likely due to a variety of factors including longer hospital stay, increased frequency of invasive procedures and pervasive antibiotic therapy. Compounding the problem is the emergence of multi-drug resistance (MDR) among many Gram+ pathogens (MRSA, S. epidermidis, Enterococcus and S. pneumoniae). Despite antibiotic stewardship and infection control, new agents against these Gram+ pathogens are urgently needed. After screening a ~60,000 preselected compound library, we obtained DNAC-2, a 4-hydroxyquinoline derivative, that exhibited antibacterial activities against MRSA and Enterococcus. We subsequently synthesized 3 series of analogues involving over 50 compounds. Two of these analogues in the 2th series, JRS-3-56 (compound 1) and JRS-4-32 (compound 2), were cidal against MRSA, S. epidermidis, E. faecalis and E. faecium, with MIC ?0.2 ?g/ml. However, both 1 and 2 have poor predicted aqueous solubility with high cLogP (7.7 and 6.0, respectively). Conversion of quinoline to quinazoline for 1 improved the cLogP (from 6.06 to 5.08) but led to a slight increase in MIC (0.25 to 2 g/ml). In the latest series, we introduced a carbonyl group at C-4 and a C to N substitution at the C-1 position, yielding compounds 3 and 4 with low cLogPs and very low MIC (0.06 g/ml for USA300), accompanied by a much tighter SAR. Using macromolecular synthesis assays, membrane-specific dye FM4- 64 and electron microscopy studies, we have evidence that 1 and 2 target the Gram+ membrane (3 and 4 also resulted in membrane defect as detected by the FM4-64 dye), but not Gram- or eukaryotic membrane, thus implying some degree of specificity. However, the exact target of these compounds which likely differs from daptomycin, is not known. In this application, we seek to define the mechanism of action of these quinoline/quinolone derivatives and further explore the SAR that governs in vitro and in vivo activities and drug disposition properties. Accordingly, we have the following specific aims: 1) design and synthesize quinoline/quinolone derivates by defining the SAR that governs activity against major Gram+ pathogens and drug disposition properties (MIC, solubility, overt toxicity and serum binding etc.); 2) delineate the mechanism of action of the quinoline/quinolone derivatives with genetic, biochemical and biophysical tools; 3) pharmacokinetic and efficacy studies where candidates compounds will be evaluated for their drug disposition properties to ensure safety and selectivity followed by selection of ?lead? compounds for full PK evaluation and efficacy studies with two animal models. The goal of these studies is to identify ?druggable membrane-active compounds? with broad Gram+ activity. If successful, we believe these compounds will represent a new class of membrane-active compounds that offer a significant advance in drug development.